Chen Bojiang, Tan Zhi, Gao Jun, Wu Wei, Liu Lida, Jin Wei, Cao Yidan, Zhao Shuang, Zhang Wen, Qiu Zhixin, Liu Dan, Mo Xianming, Li Weimin
Department of Respiratory and Critical Care Medicine, West China Hospital of Sichuan University, No. 37, Guo Xue Street, Chengdu, Sichuan, 610041, China.
Inspectiong and Quarantine Technical Center of Sichuan Entry-Exit Inspection and Quarantine Bureau, Chengdu, China.
J Exp Clin Cancer Res. 2015 Oct 21;34:126. doi: 10.1186/s13046-015-0239-1.
Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is involved in multiple cellular bioactivities. However, its clinicopathological significance in non-small cell lung cancer (NSCLC) is poorly understood.
Expressions of total rpS6 (t-rpS6) and phosphorylated rpS6 (Ser235/236, p-rpS6) were detected immunohistochemically in 316 NSCLC tissues and 82 adjacent controls, followed by statistical evaluation of the relationship between proteins expressions and patients' survivals to identify their prognostic values. Cytological experiments with overexpressing or silencing rpS6 by lentivirus in human bronchial epithelial (HBE) and NSCLC cell lines were performed to explore potential mechanisms by which rpS6 affects the clinical development of NSCLC. Additionally, specific RNA interference for Akt1, Akt2, Akt3, Akt inhibitor and subsequent cellular bioactivity tests were employed as well to investigate the upstream regulation of rpS6.
Positive rates of t-rpS6 and p-rpS6 were both significantly increased in NSCLC tissues, compared with controls (82.91 vs 62.20 % for t-rpS6; 52.22 vs 21.95 % for p-rpS6; both P < 0.001). However, only hyperphosphorylation of rpS6, expressed as either elevated p-rpS6 alone or the ratio of p-rpS6 to t-rpS6 (p-rpS6/t-rpS6) no less than 0.67, was greatly associated with the unfavorable survival of NSCLC patients, especially for cases at stage I (all P < 0.001). The independent adverse prognostic value of hyperphosphorylated rpS6 was confirmed by multivariate Cox regression analysis (hazard ratios for elevated p-rpS6 alone and p-rpS6/t-rpS6 no less than 0.67 were 2.403, 4.311 respectively, both P < 0.001). Overexpression or knockdown of rpS6, along with parallel alterations of p-rpS6, led to increased or decreased cells proliferations respectively, which were dependent on redistributions of cell cycles (all P < 0.05). Cells migration and invasion also changed with rpS6 interference (all P < 0.05). Furthermore, upstream overexpression or knockdown of Akt2 or Akt2 phosphorylation inhibition, rather than Akt1 or Akt3, resulted in striking hyperphosphorylation or dephosphorylation of mTOR, p70S6K and rpS6 (all P < 0.05), without any change in total proteins expressions. Further tests showed markedly accompanied variation of cells proliferation, cell cycle distribution and invasion (all P < 0.05).
Hyperphosphorylation of rpS6, probably regulated by the Akt2/mTOR/p70S6K signaling pathway, is closely relevant to the progression of NSCLC and it might be served as a promising therapeutic target for NSCLC treatment.
核糖体蛋白S6（rpS6）是40S核糖体亚基的组成部分，参与多种细胞生物活性。然而，其在非小细胞肺癌（NSCLC）中的临床病理意义尚不清楚。
采用免疫组织化学方法检测316例NSCLC组织和82例癌旁对照组织中总rpS6（t-rpS6）和磷酸化rpS6（Ser235/236，p-rpS6）的表达，随后对蛋白表达与患者生存率之间的关系进行统计学评估，以确定其预后价值。通过慢病毒在人支气管上皮（HBE）和NSCLC细胞系中过表达或沉默rpS6进行细胞学实验，以探讨rpS6影响NSCLC临床进展的潜在机制。此外，还采用对Akt1、Akt2、Akt3、Akt抑制剂的特异性RNA干扰及随后的细胞生物活性测试来研究rpS6的上游调控。
与对照组相比，NSCLC组织中t-rpS6和p-rpS6的阳性率均显著升高（t-rpS6分别为82.91%对62.20%；p-rpS6分别为52.22%对21.95%；均P<0.001）。然而，只有rpS6的过度磷酸化，单独表现为p-rpS6升高或p-rpS6与t-rpS6的比值（p-rpS6/t-rpS6）不低于0.67，与NSCLC患者的不良生存密切相关，尤其是I期病例（均P<0.001）。多因素Cox回归分析证实了rpS6过度磷酸化的独立不良预后价值（单独p-rpS6升高和p-rpS6/t-rpS6不低于0.67的风险比分别为2.403、4.311，均P<0.001）。rpS6的过表达或敲低，以及p-rpS6的平行变化，分别导致细胞增殖增加或减少，这取决于细胞周期的重新分布（均P<0.05）。细胞迁移和侵袭也随rpS6干扰而改变（均P<0.05）。此外，Akt2的上游过表达或敲低或Akt2磷酸化抑制，而非Akt1或Akt3，导致mTOR、p70S6K和rpS6的显著过度磷酸化或去磷酸化（均P<0.05），而总蛋白表达无任何变化。进一步测试显示细胞增殖、细胞周期分布和侵袭明显伴随变化（均P<0.05）。
rpS6的过度磷酸化可能受Akt2/mTOR/p70S6K信号通路调控，与NSCLC的进展密切相关，可能是NSCLC治疗的一个有前景的治疗靶点。
